Vol. 52, No. 1, 2009 - Alpha Chi

DNA Va c c i n e s f o r HIV
electroporation to asses plasmid expression. 12 In addition to increasing transfection rates,
there is evidence that electroporation may induce a localized inflammatory response and
therefore, a more potent immune response by trafficking immune cells to the point of
injection. 8,12 However, electroporation is not the only optimization method being examined.
For example, intradermal/subcutaneous (ID) influenza immunizations have shown to
produce significant increases in both arms of immune responses in comparison to IM. 8 The
increased immunological potency of this method is predominantly attributed to the large
density of Langerhans Cells (LCs) and dermal DCs, which are professional APCs found in
the dermal layer of the skin. Though this route of immunization is relatively painless, the ID
space can limit the volume of DNA formulation administered.
To induce the desired Th1-polarized immune response many have shown the efficacy
of molecular adjuvants. 8 These molecular adjutants serve as additional signaling molecules,
such as those secreted by DCs during T-cell activation, that are able to select the appropriate
immune response to the antigen. 8,10-13 In particular, interleukin-12 (IL-12), a cytokine
produced by APCs, has shown to preferentially activate the Th1 pathway and stimulate
considerable increases in IFNγ secretion by T-cells, NK cells and APCs. 13
Our goals for this experiment were twofold. We first assessed the ability of electroporation
to enhance transfection and expression after ID+EP using a pig model with gene constructs
expressing gfp. Secondly, we assessed the immunogenicity of optimized HIV plasmids
expressing gag and env antigens and plasmid-encoded rhesus IL-12 when delivered by ID
injection.
In this experiment we have used microelectrodes (µEP) to prevent skin damage or
discomfort during plasmid administration, with a good area of plasmid distribution, and
stimulation of both humoral and cellular responses. Electroporation conditions from µEP, as
well as plasmid doses were assessed primarily using the pig studies. For the second aspect
of our studies, rhesus macaques were immunized twice with a low dose of DNA (0.2mg/
antigen) and then immunized three times with a high dose of DNA (1.0mg/antigen) by ID
injection followed by electroporation.
Materials and Methods
Immunization protocol
In this study six Indian rhesus macaques (Macaca mulatta) were anesthetized and
subsequently immunized into 2 ID injection sites with HIV DNA vaccines containing
each of two HIV antigens (gag and env) and IL-12 expressing plasmid as an adjuvant.
Immunization of three of these animals was followed by in vivo electroporation (ID+EP).
The animals were immunized five times at weeks 0, 4, 8, 12, 16. Blood was collected every
two weeks and ELISpot assays were performed two weeks after each immunization, while
ELISA assays were performed four weeks after each immunization (Figure 3). 12
43